The tear film is necessary for the quality of the visual image and for protection of the ocular surface. Dry eye states are major causes of ocular morbidity, and deficiencies of the aqueous component of the tear film, which is produced by the lacrimal glands, have been implicated in a large proportion of dry eye cases. Little has been known about the normal mechanisms of lacrimal secretory function. In part because specific functional defects have not yet been identified, current dry eye treatments must rely on replacement with artificial tears. The proposed studies will characterize a recently discovered aspect of lacrimal acinar cell function, stimulation-associated redistribution of the sodium pump enzyme, Na,K-ATPase. In the lacrimal acinar cell, Na,K-ATPase, Na/H antiporters, Cl/HCO3 antiporters, and muscarinic acetylcholine receptors are present in the basal-lateral membranes, and in at least two fundamentally different kinds of cytoplasmic pools, the first relatively richer in the basal-lateral membrane-expressed constituents other than Na,K-ATPase, and the second relatively richer in Na,K-ATPase. Following activation of muscarininc receptors, Na,K-ATPase and other constituents are mobilized from the cytoplasmic pools and translocated to the basal-lateral membranes. Available data are consistent with the hypothesis that this overall translocation procedes in two steps: from the first cytoplasmic pool to the basal-lateral membrane, and from the second pool to the first. Experiments are proposed to identify signals for Na,K-ATPase translocation, to delineate the sequence of steps involved, to identify the functional properties of the cytoplasmic pools of Na,K-ATPase, and to clarify the relationship between these translocation phenomena and basal-lateral membrane recycling. The experimental design will utilize intact acini isolated from rat exorbital lacrimal glands. These structures retain cell:cell junctions and normal apical vs basal-lateral polarity. Because the acini can be maintained in short-term culture for up to 2 days, it will be possible to perform metabolic-, fluid phase-, and surface- labeling procedures, then subect the acini to a multidimensional subcellular fractionation procedure, incorporating differential sedimentation, density gradient centrifugation, and partitioning in aqueous polymer phase systems, which resolves a series of subcellular membrane populations. At the same time, it will be possible to correlate morphological and immunocytochemical results with the analytical fractionation data.